RESUMEN

OBJECTIVES: The aim of this study was to noninvasively detect the anti-inflammatory properties of the novel liver X receptor agonist R211945. BACKGROUND: R211945 induces reversal cholesterol transport and modulates inflammation in atherosclerotic plaques. We aimed to characterize with (18)F-fluorodeoxyglucose (FDG)-positron emission tomography (PET)/computed tomography (CT) and dynamic contrast-enhanced cardiac magnetic resonance (DCE-CMR) inflammation and neovascularization, respectively, in atherosclerotic plaques with R211945 treatment compared with atorvastatin treatment and a control. METHODS: Twenty-one atherosclerotic New Zealand white rabbits were divided into 3 groups (control, R211945 [3 mg/kg orally], and atorvastatin [3 mg/kg orally] groups). All groups underwent (18)F-FDG-PET/CT and DCE-CMR at baseline and at 1 and 3 months after treatment initiation. Concomitantly, serum metabolic parameters and histology were assessed. For statistical analysis, continuous DCE-CMR and PET/CT outcomes were modeled as linear functions of time by using a linear mixed model, whereas the histological data, animal characteristics data, and nonlinear regression imaging data were analyzed with a 2-tailed Student t test. RESULTS: (18)F-FDG-PET/CT detected a decrease in mean and maximum standard uptake values (SUV) over time in the R211945 group (both p = 0.001), indicating inflammation regression. The atorvastatin group displayed no significant change (p = 0.371 and p = 0.600, respectively), indicating no progression or regression. The control group demonstrated an increase in SUV (p = 0.01 and p = 0.04, respectively), indicating progression. There was a significant interaction between time and group for mean and maximum SUV (p = 0.0003 and p = 0.0016, respectively) . DCE-CMR detected a trend toward difference (p = 0.06) in the area under the curve in the atorvastatin group, suggesting a decrease in neovascularization. There was no significant interaction between time and group (p = 0.6350 and p = 0.8011, respectively). Macrophage and apolipoprotein B immunoreactivity decreased in the R211945 and atorvastatin groups (p < 0.0001 and p = 0.0004, respectively), and R211945 decreased oxidized phospholipid immunoreactivity (p = 0.02). CONCLUSIONS: Noninvasive imaging with (18)F-FDG-PET/CT and DCE-CMR and histological analysis demonstrated significant anti-inflammatory effects of the LXR agonist R211945 compared with atorvastatin. The results suggest a possible role for LXR agonists in the treatment of atherosclerosis.

Asunto(s)

Ácidos Heptanoicos/uso terapéutico , Inhibidores de Hidroximetilglutaril-CoA Reductasas/uso terapéutico , Receptores Nucleares Huérfanos/agonistas , Placa Aterosclerótica/tratamiento farmacológico , Pirroles/uso terapéutico , Animales , Apolipoproteínas B/metabolismo , Atorvastatina , Progresión de la Enfermedad , Fluorodesoxiglucosa F18 , Inmunohistoquímica , Receptores X del Hígado , Macrófagos/metabolismo , Imagen Multimodal , Placa Aterosclerótica/metabolismo , Tomografía de Emisión de Positrones , Conejos , Radiofármacos , Tomografía Computarizada por Rayos X , Resultado del Tratamiento

Asunto(s)

Diseño de Fármacos , Hipolipemiantes , 1-Alquil-2-acetilglicerofosfocolina Esterasa/antagonistas & inhibidores , Animales , Apolipoproteína A-I/sangre , Proteínas Portadoras/antagonistas & inhibidores , Proteínas de Transferencia de Ésteres de Colesterol/antagonistas & inhibidores , Enfermedad Coronaria/etiología , Enfermedad Coronaria/prevención & control , Modelos Animales de Enfermedad , Farnesil Difosfato Farnesil Transferasa/antagonistas & inhibidores , Humanos , Hiperlipidemias/tratamiento farmacológico , Hiperlipidemias/etiología , Oligorribonucleótidos Antisentido , Probucol/análogos & derivados , Receptores Acoplados a Proteínas G/agonistas , Receptores Nicotínicos

RESUMEN

The objective of the present study was to determine whether a novel acyl-CoA:cholesterol acyltransferase (ACAT) inhibitor, pactimibe sulfate (CS-505), could reduce atherosclerotic lesions beyond and independent of the reduction achieved by cholesterol lowering alone from two different types of lesions. (1) Early lesion model. Twelve-week-old apolipoprotein E (apoE)(-/-) mice were treated with 0.03 or 0.1% (w/w) CS-505, 0.1 or 0.3% avasimibe (CI-1011), or 3% cholestyramine for 12 weeks. Each treatment significantly reduced plasma cholesterol by a similar degree (43-48%). The antiatherosclerotic activity of 0.1% CS-505, however, was more efficacious than the effects of the other treatments (90% versus 40-50%). (2) Advanced lesion model. Twenty-four-week-old apoE(-/-) mice were treated with 0.03 or 0.1% CS-505 or 0.1% CI-1011 for 12 weeks. CS-505 at 0.1% revealed enhanced lesion reduction compared with 0.1% CI-1011 (77% versus 54%), whereas the plasma cholesterol-lowering effect of 0.1% CS-505 was almost the same as that of 0.1% CI-1011. Furthermore, immunohistochemical analysis demonstrated that CS-505 significantly reduced the number of macrophages and expression of matrix metalloproteinase (MMP)-2, MMP-9, and MMP-13. These data indicate that CS-505 can reduce and stabilize atherosclerotic lesions. This antiatherosclerotic activity is exerted via both cholesterol lowering and direct ACAT inhibition in plaque macrophages.

Asunto(s)

Anticolesterolemiantes/uso terapéutico , Apolipoproteínas E/deficiencia , Aterosclerosis/tratamiento farmacológico , Ácidos Indolacéticos/uso terapéutico , Esterol O-Aciltransferasa/antagonistas & inhibidores , Animales , Anticolesterolemiantes/farmacología , Aterosclerosis/metabolismo , Aterosclerosis/patología , Línea Celular , Humanos , Ácidos Indolacéticos/farmacología , Lípidos/sangre , Ratones , Ratones Noqueados , Monocitos/efectos de los fármacos

RESUMEN

Pactimibe sulfate, [7-(2,2-dimethylpropanamido)-4,6-dimethyl-1-octylindolin-5-yl]acetic acid hemisulfate, a novel Acyl-coenzyme A:cholesterol acyltransferase (ACAT) inhibitor, was investigated in vitro and in vivo to characterize its potential. Pactimibe exhibited dual inhibition for ACAT1 and ACAT2 (concentrations inhibiting 50% [IC50s] at micromolar levels) more potently than avasimibe. Kinetic analysis revealed pactimibe is a noncompetitive inhibitor of oleoyl-CoA (Ki value: 5.6 microM). Furthermore, pactimibe markedly inhibited cholesteryl ester formation (IC50: 6.7 microM) in human monocyte-derived macrophages, and inhibited copper-induced oxidation of low density lipoprotein more potently than probucol. Pactimibe exerted potent lipid-lowering and anti-atherosclerotic effects in atherogenic diet-fed hamsters. At doses of 3 and 10 mg/kg for 90 days, pactimibe decreased serum total cholesterol by 70% and 72%, and aortic fatty streak area by 79% and 95%, respectively. Despite similar cholesterol lowering, fatty streak area reduction was greater by 10 mg/kg. These results suggest that ACAT1/2 dual inhibitor pactimibe has anti-atherosclerotic potential beyond its plasma cholesterol-lowering activity.

Asunto(s)

Arteriosclerosis/prevención & control , Ácidos Indolacéticos/farmacología , Esterol O-Aciltransferasa/antagonistas & inhibidores , Acilcoenzima A/metabolismo , Animales , Catálisis/efectos de los fármacos , Línea Celular , Sistema Libre de Células/enzimología , Sistema Libre de Células/metabolismo , Ésteres del Colesterol/metabolismo , Cricetinae , Dieta Aterogénica , Relación Dosis-Respuesta a Droga , Humanos , Hipercolesterolemia/clasificación , Hipercolesterolemia/etiología , Hipercolesterolemia/prevención & control , Hipolipemiantes/química , Hipolipemiantes/farmacología , Ácidos Indolacéticos/química , Lípidos/sangre , Lipoproteínas LDL/genética , Macrófagos/citología , Macrófagos/efectos de los fármacos , Macrófagos/metabolismo , Monocitos/citología , Monocitos/efectos de los fármacos , Monocitos/metabolismo , Oxidación-Reducción/efectos de los fármacos , Fosfatidilcolina-Esterol O-Aciltransferasa/antagonistas & inhibidores , Fosfatidilcolina-Esterol O-Aciltransferasa/metabolismo , Conejos , Ratas , Ratas Sprague-Dawley , Esterol O-Aciltransferasa/metabolismo , Esterol O-Aciltransferasa 2

RESUMEN

Fluid shear stress generated by blood flowing over the endothelium is a major determinant of arterial tone, vascular remodeling, and atherogenesis. Nitric oxide (NO) produced by endothelial NO synthase (eNOS) plays an essential role in regulation of vascular function and structure by blood flow. Although cyclosporin A (CsA), an inhibitory ligand of cyclophilin A, is a widely used immunosuppressive drug, it causes arterial hypertension in part by impairing eNOS-dependent vasodilation. Here we show that CsA inhibits fluid shear stress-mediated eNOS activation in endothelial cells via decreasing cholesterol content in caveolae. Exposure of cultured bovine aortic endothelial cells to 1 mum CsA for 1 h significantly inhibited NO production and eNOS phosphorylation at Ser-1179 induced by flow (shear stress=dynes/cm2). The effect of CsA was not related to inhibition of two known eNOS kinases, protein kinase B (Akt) and protein kinase A, because CsA did not affect Akt or protein kinase A activation. In rabbit aorta perfused ex vivo, CsA also significantly inhibited flow-induced eNOS phosphorylation at Ser-1179 but had no effect on Akt measured by phosphorylation at Ser-473. However, CsA treatment decreased cholesterol content in caveolae and displaced eNOS from caveolae, which may be caused by CsA disrupting the association of caveolin-1 and cyclophilin A. The magnitude of the cholesterol depleting effect was similar to that of beta-cyclodextrin, a cholesterol-binding molecule, and beta-cyclodextrin had a similar inhibitory effect on flow-mediated eNOS activation. Treating bovine aortic endothelial cells for 24 h with 30 mug/ml cholesterol blocked the CsA effect and restored eNOS phosphorylation in response to flow. These data suggest that decreasing cholesterol content in caveolae by CsA is a potentially important pathogenic mechanism for CsA-induced endothelial dysfunction and hypertension.

Asunto(s)

Colesterol/metabolismo , Ciclosporina/farmacología , Inmunosupresores/farmacología , Óxido Nítrico Sintasa/metabolismo , Adenoviridae/genética , Animales , Aorta/metabolismo , Western Blotting , Bovinos , Membrana Celular/metabolismo , Células Cultivadas , Proteínas Quinasas Dependientes de AMP Cíclico/química , Detergentes/farmacología , Endotelio Vascular/enzimología , Endotelio Vascular/metabolismo , Activación Enzimática , Immunoblotting , Cinética , Óxido Nítrico/metabolismo , Óxido Nítrico Sintasa de Tipo III , Fosforilación , Proteínas Serina-Treonina Quinasas/química , Proteínas Proto-Oncogénicas/química , Proteínas Proto-Oncogénicas c-akt , Conejos , Serina/química , Estrés Mecánico , Factores de Tiempo , beta-Ciclodextrinas/farmacología

RESUMEN

Sphingosine 1-phosphate (S1P) is a bioactive lipid generated during vascular injury that regulates cell growth, differentiation, survival, and motility via endothelial differentiation gene (EDG) family G protein-coupled receptors. Although several G protein-coupled receptor ligands transactivate receptor tyrosine kinases, such as the epidermal growth factor receptor (EGFR), S1P-stimulated receptor tyrosine kinase transactivation has not been well studied. We show that platelet-derived growth factor beta receptor (PDGFbetaR) and EGFR are tyrosine phosphorylated in response to S1P in rat aortic vascular smooth muscle cells (VSMCs). S1P-stimulated transactivation of PDGFbetaR and EGFR was mediated via Gi-coupled EDG receptors. S1P-stimulated transactivation of EGFR and PDGFbetaR was dependent on Src, reactive oxygen species, and cholesterol-rich membranes. A phosphoinositide 3-kinase-Akt pathway was activated by S1P and blocked by AG1296 and AG1478. Activation of extracellular signal-regulated kinase (ERK) 1 and ERK2 pathway by S1P was blocked only by AG1478. In Chinese hamster ovary cells that expressed exogenous EDG-1, activation of Akt and ERK1/2 in response to S1P was observed and was enhanced by coexpression of PDGFbetaR or EGFR. S1P-mediated VSMC proliferation was shown to be secondary to transactivation, because it was suppressed by AG1296 and AG1478. These data establish S1P as an important stimulus for EGFR and PDGFbetaR activation in VSMCs that may have important implications for the vessel response to injury.

Asunto(s)

Receptores ErbB/biosíntesis , Lisofosfolípidos/farmacología , Músculo Liso Vascular/citología , Miocitos del Músculo Liso/efectos de los fármacos , Receptor beta de Factor de Crecimiento Derivado de Plaquetas/biosíntesis , Esfingosina/farmacología , Activación Transcripcional/efectos de los fármacos , Animales , Aorta , Células CHO , Cricetinae , Cricetulus , Activación Enzimática/efectos de los fármacos , Inhibidores Enzimáticos/farmacología , Receptores ErbB/genética , Subunidades alfa de la Proteína de Unión al GTP Gi-Go/fisiología , Sistema de Señalización de MAP Quinasas/efectos de los fármacos , Lípidos de la Membrana/fisiología , Músculo Liso Vascular/metabolismo , Miocitos del Músculo Liso/metabolismo , Fosfatidilinositol 3-Quinasas/fisiología , Fosforilación/efectos de los fármacos , Procesamiento Proteico-Postraduccional/efectos de los fármacos , Proteínas Serina-Treonina Quinasas/fisiología , Proteínas Proto-Oncogénicas/fisiología , Proteínas Proto-Oncogénicas c-akt , Quinazolinas , Ratas , Ratas Sprague-Dawley , Receptor beta de Factor de Crecimiento Derivado de Plaquetas/genética , Esfingosina/análogos & derivados , Superóxidos/metabolismo , Tirfostinos/farmacología

RESUMEN

Fluid shear stress generated by blood flowing over the endothelium is a major determinant of arterial tone, vascular remodeling, and atherogenesis. Nitric oxide (NO) produced by endothelial NO synthase (eNOS) plays an essential role in regulation of vascular function and structure by blood flow, but the molecular mechanisms that transduce mechanical force to eNOS activation are not well understood. In this study, we found that laminar flow (shear stress=12 dyne/cm2) rapidly activates vascular endothelial growth factor receptor 2 (VEGFR2) in a ligand-independent manner and leads to eNOS activation in cultured endothelial cells. Flow-stimulated VEGFR2 recruits phosphoinositide 3-kinase and mediates activation of Akt and eNOS. Inhibiting VEGFR2 kinase with selective inhibitors blocks flow-induced activation of Akt and eNOS and production of NO. Decreasing VEGFR2 expression with antisense VEGFR2 oligonucleotides significantly attenuates activation of Akt and eNOS. Furthermore, Src kinases are involved in flow-stimulated VEGFR2 because inhibiting Src kinases by PP2, a selective inhibitor for Src kinases, abolishes flow-induced VEGFR2 tyrosine phosphorylation and downstream signaling. Finally, we show that inhibiting VEGFR2 kinase significantly reduces flow-mediated NO-dependent arteriolar dilation in vivo. These data identify VEGFR2 as a key mechanotransducer that activates eNOS in response to blood flow.

Asunto(s)

Endotelio Vascular/metabolismo , Óxido Nítrico Sintasa/metabolismo , Proteínas Serina-Treonina Quinasas , Receptor 2 de Factores de Crecimiento Endotelial Vascular/metabolismo , Animales , Anticuerpos Monoclonales/farmacología , Benzoquinonas , Vasos Sanguíneos/efectos de los fármacos , Vasos Sanguíneos/fisiología , Línea Celular , Mejilla/irrigación sanguínea , Cricetinae , Endotelio Vascular/citología , Endotelio Vascular/enzimología , Activación Enzimática , Inhibidores Enzimáticos/farmacología , Genisteína/farmacología , Integrina alfaVbeta3/inmunología , Lactamas Macrocíclicas , Ligandos , Óxido Nítrico/biosíntesis , Fosfatidilinositol 3-Quinasas/metabolismo , Fosforilación/efectos de los fármacos , Proteínas Proto-Oncogénicas/metabolismo , Proteínas Proto-Oncogénicas c-akt , Quinonas/farmacología , Rifabutina/análogos & derivados , Transducción de Señal/efectos de los fármacos , Estrés Mecánico , Receptor 2 de Factores de Crecimiento Endotelial Vascular/antagonistas & inhibidores , Receptor 2 de Factores de Crecimiento Endotelial Vascular/genética , Vasodilatación/efectos de los fármacos , Familia-src Quinasas/metabolismo

RESUMEN

c-Jun NH2-terminal kinase (JNK) is activated by a number of cellular stimuli including reactive oxygen species (ROS). Previous studies have demonstrated that fluid shear stress (flow) inhibits cytokine-induced JNK activation in endothelial cells (ECs). In the present study, we show JNK activation by ROS in ECs and hypothesized that flow inhibits ROS-induced JNK activation in ECs via modulation of cellular protection systems against ROS. JNK was activated by 300 micro mol/L hydrogen peroxide (H2O2) in bovine lung microvascular ECs (BLMVECs) with a peak at 60 minutes after stimulation (6.3+/-1.2-fold increase). Preexposure of BLMVECs to physiological steady laminar flow (shear stress=12 dyne/cm2) for 10 minutes significantly decreased H2O2-induced JNK activation. Thioredoxin and glutathione are cellular antioxidants that protect cells against ROS. Flow induced a significant increase in the ratio of reduced glutathione to oxidized glutathione consistent with a 1.6-fold increase in glutathione reductase (GR) activity. Preincubation of BLMVECs with the GR inhibitor, 1,3 bis-(2 chloroethyl)-1-nitrosourea, abolished the inhibitory effect of flow. In contrast, preincubation of BLMVECs with azelaic acid, a specific inhibitor for thioredoxin reductase, did not alter the effect of flow on H2O2-induced JNK activation. Overexpression of GR mimicked the effect of flow to inhibit JNK activation. These results suggest that flow activates GR, an important regulator of the intracellular redox state of glutathione, and exerts a protective mechanism against oxidative stress in endothelial cells.

Asunto(s)

Endotelio Vascular/enzimología , Glutatión Reductasa/fisiología , Peróxido de Hidrógeno/antagonistas & inhibidores , Proteínas Quinasas Activadas por Mitógenos/metabolismo , Oxidantes/antagonistas & inhibidores , Animales , Bovinos , Células Cultivadas , Relación Dosis-Respuesta a Droga , Endotelio Vascular/efectos de los fármacos , Endotelio Vascular/metabolismo , Activación Enzimática , Glutatión/fisiología , Glutatión Reductasa/genética , Proteínas Quinasas JNK Activadas por Mitógenos , Cinética , Modelos Químicos , Oxidación-Reducción , Estrés Oxidativo , Transducción de Señal , Estrés Mecánico , Tiorredoxinas/metabolismo , Transfección

RESUMEN

Sphingosine 1-phosphate (S1P) and vascular endothelial growth factor (VEGF) elicit numerous biological responses including cell survival, growth, migration, and differentiation in endothelial cells mediated by the endothelial differentiation gene, a family of G-protein-coupled receptors, and fetal liver kinase-1/kinase-insert domain-containing receptor (Flk-1/KDR), one of VEGF receptors, respectively. Recently, it was reported that S1P or VEGF treatment of endothelial cells leads to phosphorylation at Ser-1179 in bovine endothelial nitric oxide synthase (eNOS), and this phosphorylation is critical for eNOS activation. S1P stimulation of eNOS phosphorylation was shown to involve G(i) protein, phosphoinositide 3-kinase, and Akt. VEGF also activates eNOS through Flk-1/KDR, phosphoinositide 3-kinase, and Akt, which suggested that S1P and VEGF may share upstream signaling mediators. We now report that S1P treatment of bovine aortic endothelial cells acutely increases the tyrosine phosphorylation of Flk-1/KDR, similar to VEGF treatment. S1P-mediated phosphorylation of Flk-1/KDR, Akt, and eNOS were all inhibited by VEGF receptor tyrosine kinase inhibitors and by antisense Flk-1/KDR oligonucleotides. Our study suggests that S1P activation of eNOS involves G(i), calcium, and Src family kinase-dependent transactivation of Flk-1/KDR. These data are the first to establish a critical role of Flk-1/KDR in S1P-stimulated eNOS phosphorylation and activation.

Asunto(s)

Factores de Crecimiento Endotelial/farmacología , Endotelio Vascular/fisiología , Péptidos y Proteínas de Señalización Intercelular/farmacología , Linfocinas/farmacología , Lisofosfolípidos , Óxido Nítrico Sintasa/metabolismo , Proteínas Serina-Treonina Quinasas , Proteínas Proto-Oncogénicas/metabolismo , Esfingosina/análogos & derivados , Esfingosina/farmacología , Receptor 2 de Factores de Crecimiento Endotelial Vascular/genética , Animales , Aorta , Secuencia de Bases , Bovinos , División Celular/efectos de los fármacos , Supervivencia Celular/efectos de los fármacos , Células Cultivadas , Endotelio Vascular/efectos de los fármacos , Activación Enzimática , Proteínas de Unión al GTP/metabolismo , Cinética , Óxido Nítrico Sintasa de Tipo III , Oligodesoxirribonucleótidos Antisentido/farmacología , Fosfatidilinositol 3-Quinasas/metabolismo , Fosforilación , Proteínas Proto-Oncogénicas c-akt , Activación Transcripcional , Factor A de Crecimiento Endotelial Vascular , Receptor 2 de Factores de Crecimiento Endotelial Vascular/metabolismo , Factores de Crecimiento Endotelial Vascular

RESUMEN

Stachybotrys sp. RF-7260 was found to produce stachyflins, novel anti-influenza virus agents, under solid-state fermentation conditions. Feeding DL-lysine to a culture of Stachybotrys sp. RF-7260 induced the formation of the novel compounds, SQ-02-S-L2 and -L1, and feeding DL-valine the formation of SQ-02-S-VI and -V2. The structures of these metabolites were determined by detailed 2D NMR analyses in comparison with acetylstachyflin. SQ-02-S-L2 and -L1 have the lysine moiety and SQ-02-S-V1 has the valine moiety. SQ-02-S-V2 has an amidine moiety instead of the lactam moiety in acetylstachyflin. SQ-02-S-L2, -L1 and -V1, substituted on the lactam amide hydrogen, displayed only a low level of the antiviral activity. However, deacetyl SQ-02-S-V2 showed potent antiviral activity similar to stachyflin.

Asunto(s)

Antivirales/química , Compuestos Heterocíclicos de 4 o más Anillos/química , Indoles/química , Sesquiterpenos/química , Stachybotrys/metabolismo , Animales , Antivirales/aislamiento & purificación , Antivirales/farmacología , Bovinos , Línea Celular , Fermentación , Compuestos Heterocíclicos de 4 o más Anillos/aislamiento & purificación , Compuestos Heterocíclicos de 4 o más Anillos/farmacología , Indoles/aislamiento & purificación , Indoles/farmacología , Virus de la Influenza A/efectos de los fármacos , Riñón/citología , Sesquiterpenos/aislamiento & purificación , Sesquiterpenos/farmacología , Relación Estructura-Actividad

RESUMEN

Grb2-associated binder-1 (Gab1) is an adapter protein related to the insulin receptor substrate family. It is a substrate for the insulin receptor as well as the epidermal growth factor (EGF) receptor and other receptor-tyrosine kinases. To investigate the role of Gab1 in signaling pathways downstream of growth factor receptors, we stimulated rat aortic vascular smooth muscle cells (VSMC) with EGF and platelet-derived growth factor (PDGF). Gab1 was tyrosine-phosphorylated by EGF and PDGF within 1 min. AG1478 (an EGF receptor kinase-specific inhibitor) failed to block PDGF-induced Gab1 tyrosine phosphorylation, suggesting that transactivated EGF receptor is not responsible for this signaling event. Because Gab1 associates with phospholipase Cgamma (PLCgamma), we studied the role of the PLCgamma pathway in Gab1 tyrosine phosphorylation. Gab1 tyrosine phosphorylation by PDGF was impaired in Chinese hamster ovary cells expressing mutant PDGFbeta receptor (Y977F/Y989F: lacking the binding site for PLCgamma). Pretreatment of VSMC with (a specific PLCgamma inhibitor) inhibited Gab1 tyrosine phosphorylation as well, indicating the importance of the PLCgamma pathway. Gab1 was tyrosine-phosphorylated by phorbol ester to the same extent as PDGF stimulation. Studies using antisense protein kinase C (PKC) oligonucleotides and specific inhibitors showed that PKCalpha and PKCepsilon are required for Gab1 tyrosine phosphorylation. Binding of Gab1 to the protein-tyrosine phosphatase SHP2 and phosphatidylinositol 3-kinase was significantly decreased by PLCgamma and/or PKC inhibition, suggesting the importance of the PLCgamma/PKC-dependent Gab1 tyrosine phosphorylation for the interaction with other signaling molecules. Because PDGF-mediated ERK activation is enhanced in Chinese hamster ovary cells that overexpress Gab1, Gab1 serves as an important link between PKC and ERK activation by PDGFbeta receptors in VSMC.

Asunto(s)

Isoenzimas/fisiología , Fosfoproteínas/metabolismo , Factor de Crecimiento Derivado de Plaquetas/farmacología , Proteína Quinasa C/fisiología , Tirosina/metabolismo , Animales , Activación Enzimática , Factor de Crecimiento Epidérmico/farmacología , Masculino , Proteínas Quinasas Activadas por Mitógenos/metabolismo , Fosfolipasa C gamma , Fosforilación , Proteína Quinasa C-alfa , Proteína Quinasa C-epsilon , Ratas , Ratas Sprague-Dawley , Fosfolipasas de Tipo C/fisiología